A separation process by liquid chromatography, which comprises sending a mixture sample in a large amount together with a liquid mobile phase to at least one column, into which a packing having a large particle size has been charged, to preliminarily fractionate the components of the mixture, taking out fractions containing objective components from the eluate from the first column, and recycling said fractions within a loop provided with at least one second column, into which a packing having a large particle size has been charged, to separate precisely the objective components.According to this process, it is possible to separate economically objective components from the sample loaded in a large amount.